The present invention relates to a method of protecting tissue from apoptosis using zinc ionophores. The present invention also relates to a method of protecting cells against the harmful effects of injurious agents, for example, oxidants, TNFxcex1, neurotoxins, ischemia and radiation.
Zinc plays a critical role in cellular biology, and is involved in virtually every important cellular process such as transcription, translation, ion transport, and others (O""Halloran, T. V. (1993) Science 261:715-725; Cousins, R. J. (1994) Annu.Rev.Nutr. 14:449-469; Harrison, N. L. et al. (1994) Neuropharmacology 33:935-952; Berg, J. M. et al. (1996) Science 271:1081-1085). The involvement of cellular zinc in apoptosis has been recognized for close to 20 years (Sunderman, F. W., Jr. (1995) Ann.Clin.Lab.Sci. 25:134-142; Fraker, P. J. et al. (1997) Proc.Soc.Exp.Biol.Med. 215:229-236.). However, the full nature of this involvement is not fully understood. Apoptosis is a form of programmed cell death normally activated under physiological conditions, such as involution in tissue remodelling during morphogenesis, and several immunological processes. The apoptotic process is characterized by cell shrinkage, chromatin condensation, and internucleosomal degradation of the cell""s DNA (Verhaegen et al. (1995) Biochem. Pharmacol. 50 (7):1021-1029).
Numerous in vitro studies have been done recently in an attempt to elucidate the role of intracellular zinc. Although some studies have suggested that zinc may actually induce apoptosis (Xu, J. et al. (1996) Am.J.Physiol. 270:G60-G70; Kim, Y. H. et al., (1999) Neuroscience 89:175-182), most have concluded that increasing the intracellular concentrations of zinc blocks apoptosis (Sunderman, F. W., Jr. (1995) Ann.Clin.Lab.Sci. 25:134-142; Adebodun, F. et al. (1995) J.Cell.Physiol. 163:80-86; Zalewski, P. D., et al. (1993) Biochem.J. 296:403-408), and that decreasing the zinc concentration promotes apoptosis (Jiang, S., et al. (1995) Lab.Invest. 73:111-117; Treves, S., et al. (1994) Exp.Cell Res. 211:339-343; Ahn, Y. H., et al. (1998) Exp.Neurol. 154:47-56). The manner in which increased intracellular zinc affords protection against apoptosis is not clear. (Truong-Tran, A. Q. et al., (2000) J. Nutr. 130:1459S-1466S) One theory proposes that zinc inactivates the intracellular endonuclease(s) responsible for apoptotic DNA fragmentation (Shiokawa, D., et al. (1994) Eur.J.Biochem. 226:23-30; Yao, M. et al., (1996) J.Mol.Cell.Cardiol. 28:95-101). Other recent studies have suggested that zinc can inhibit caspases (Jiang, S., et al. (1997) Cell Death Differ. 4:39-50; Perry, D. K., et al. (1997) J.Biol.Chem. 272:18530-18533; Maret, W., et al. (1999) Proc.Natl.Acad.Sci.USA 96:1936-1940), or block the activation of caspases (Aiuchi, T., et al. (1998) J.Biochem. 124:300-303). However, in view of the large number of intracellular roles played by zinc, it seems likely that its anti-apoptotic mechanisms may be more complex, possibly involving gene expression and cellular signalling pathways. In fact, recent studies support a role for zinc transients in intracellular signalling and gene expression (O""Halloran, T. V. (1993) Science 261:715-725; Berg, J. M., et al., (1996) Science 271:1081-1085).
In contrast to the large number of in vitro studies, very few studies have attempted to examine the protective effects of zinc in vivo. It is important to note that most of the studies that have explored this possibility have focused on the pretreatment of tissues with zinc prior to injury. Using this approach, a number of studies have demonstrated that pretreatment of animals with zinc at least 24 hours prior to injury provided some measure of protection against apoptosis (Thomas, D. J. et al., (1991) Toxicology 68:327-337; Matsushita, K., et al., (1996) Brain Res. 743:362-365; Klosterhalfen, B., et al., (1997) Shock 7:254-262), presumably as a result of the well established ability of zinc to boost the immune system (Cunningham-Rundles, S., et al., (1990) Ann.N.Y.Acad.Sci. 587:113-122). Also, one study showed that several days of zinc dietary supplementation concomitant with i.p. injection of carbon tetrachloride protected against liver apoptosis (Cabre, M. et al. (1999) J. Hepatol. 31:228-234). However, no studies have demonstrated the efficacy of zinc when administered acutely and post-injury, a much more clinically relevant setting.
Zinc-pyrithione (zinc pyridinethione, C10H8N2O2S2Zn, MW 317.75, commercially available from Sigma) is the active ingredient in the anti-dandruff shampoo Head and Shoulders(copyright) (U.S. Pat. Nos. 3,236,733, and 3,281,366, both 1966), as well as a number of other topical skin treatment formulations. It is a fungicide and bactericide at high concentrations. It is highly lipophilic and therefore penetrates membranes easily. This permits zinc pyrithione to transport zinc across cell membranes, thereby conferring on this compound (i.e. zinc pyrithione) the properties of a zinc ionophore. The anti-apoptotic effect of zinc pyrithione was first observed in vitro by Zalewski and coworkers, who showed that micromolar concentrations of this compound protected lymphocytic leukemia cells against colchicine-induced apoptosis (Giannakis, C., et al. (1991) Biochem. Biophys. Res. Commun. 181:915-920). The rationale for the use of this zinc ionophore was to facilitate the transport of Zn2+ into the target cells. This is necessitated by the fact that all eukaryotic cells strictly regulate the membrane transport of Zn2+, making it very difficult to modulate the intracellular concentration and distribution of Zn2+. Zalewski""s group has since published a number of other studies, all of them in vitro, confirming the ability of micromolar concentrations of zinc-pyrithione to rapidly transport Zn2+ into cells and to thereby prevent apoptosis (Zalewski, P. D., et al. (1994) supra; Zalewski, P. D., et al. (1993) Biochem. J. 296:403-408). One confirmatory study, also in vitro, has been published from another laboratory (Tempel, K.-H. et al., (1993) Arch.Toxicol. 67:318-324).
In addition to zinc-pyrithione, another group of zinc ionophores, the dithiocarbamates, has been shown to affect apoptosis in vitro. (Orrenius, S. et al. (1996) Biochem. Soc. Trans. 24:1032-1038; Stefan, C. et al. (1997) Chem. Res. Toxicol. 10:636-643; Erl, W. et al., (2000) Am. J. Physiol. 278:C1116-C1125). However, no attempts have been made to examine in vivo the protective effects of zinc-pyrithione, zinc-bound dithiocarbamates or any other known zinc ionophore at nanomolar or picomolar concentrations.
The ability of zinc-pyrithione and zinc-diethyldithiocarbamates to protect tissue against apoptosis in three models of in vivo injury, as well as two in vitro models is presented. In each case a pronounced anti-apoptotic effect was achieved. The ability of zinc-pyrithione, zinc diethyldithiocarbamates and zinc 5,7-diodo-8-hydroxyquinoline to treat and prevent seizures is also provided.
Thus, according to the present invention there is provided a method to protect against apoptosis and treat seizures using one or more zinc ionophores.
In one embodiment of the present invention there is provided a method of treating apoptosis in mammalian tissue such as heart, brain, and eye tissue by administering to a mammalian subject in need thereof a pharmaceutically effective amount of at least one zinc ionophore.
In another embodiment of the present invention there is provided a pharmaceutical composition comprising a zinc ionophore and a pharmaceutically acceptable carrier.
In a further embodiment of the present invention there is provided a method of protecting against the harmful effects of injurious agents such as TNFxcex1, neurotoxins, ischemia and radiation by administering to a subject in need of such protection an effective amount of a zinc ionophore.
In a still further embodiment of the present invention there is provided a method of treating or preventing seizures by administering to a subject in need thereof a pharmaceutically effective amount of at least one zinc ionophore.